WO2016076231A1 - Procédé de production de lipide à l'aide d'acyl-acp thioestérase - Google Patents

Procédé de production de lipide à l'aide d'acyl-acp thioestérase Download PDF

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WO2016076231A1
WO2016076231A1 PCT/JP2015/081356 JP2015081356W WO2016076231A1 WO 2016076231 A1 WO2016076231 A1 WO 2016076231A1 JP 2015081356 W JP2015081356 W JP 2015081356W WO 2016076231 A1 WO2016076231 A1 WO 2016076231A1
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protein
amino acid
acid sequence
seq
acyl
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PCT/JP2015/081356
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Japanese (ja)
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達郎 尾崎
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花王株式会社
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Priority to AU2015347886A priority Critical patent/AU2015347886B2/en
Priority to MYPI2017000570A priority patent/MY182519A/en
Priority to US15/520,138 priority patent/US10280440B2/en
Priority to JP2016559023A priority patent/JP6629749B2/ja
Publication of WO2016076231A1 publication Critical patent/WO2016076231A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/02—Thioester hydrolases (3.1.2)
    • C12Y301/02014—Oleoyl-[acyl-carrier-protein] hydrolase (3.1.2.14), i.e. ACP-thioesterase

Definitions

  • the present invention relates to a method for producing a lipid using acyl-ACP thioesterase.
  • the present invention also relates to an acyl-ACP thioesterase used in the method, a gene encoding the same, and a transformant into which the gene has been introduced.
  • Fatty acids are one of the main constituents of lipids, and constitute lipids such as triacylglycerol produced by ester bonds with glycerin in vivo. In many animals and plants, fatty acids are also stored and used as energy sources. Fatty acids and lipids stored in animals and plants are widely used for food or industry. For example, derivatives of higher alcohols obtained by reducing higher fatty acids having about 12 to 18 carbon atoms are used as surfactants. Alkyl sulfate esters and alkylbenzene sulfonates are used as anionic surfactants. Polyoxyalkylene alkyl ethers, alkyl polyglycosides, and the like are used as nonionic surfactants.
  • surfactants are used as cleaning agents or disinfectants.
  • Alkylamine salts and mono- or dialkyl quaternary amine salts as derivatives of the same higher alcohol are routinely used for fiber treatment agents, hair rinse agents, disinfectants, and the like.
  • Benzalkonium-type quaternary ammonium salts are routinely used as bactericides and preservatives.
  • higher alcohols having about 18 carbon atoms are useful as plant growth promoters.
  • fatty acids and lipids are widely used, and therefore, attempts have been made to improve the productivity of fatty acids and lipids in vivo in plants and the like. Furthermore, since the use and usefulness of fatty acids depend on the number of carbon atoms, attempts have been made to control the number of carbon atoms of fatty acids, that is, the chain length. For example, a method of accumulating fatty acids having 12 carbon atoms by introducing acyl-ACP thioesterase derived from bay ( Umbellularia californica (California bay)) has been proposed (Patent Document 1, Non-Patent Document 1).
  • the present invention relates to a method for producing lipid, in which a transformant in which a gene encoding any one of the following proteins (A) to (C) is introduced into a host and lipid is collected from the culture.
  • a protein comprising the amino acid sequence of positions 91 to 348 of SEQ ID NO: 1.
  • B A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 and having acyl-ACP thioesterase activity.
  • C A protein having the amino acid sequence of the protein (A) or (B) and having acyl-ACP thioesterase activity.
  • the present invention also relates to the proteins (A) to (C) (hereinafter also referred to as the protein of the present invention or acyl-ACP thioesterase).
  • the present invention also relates to a gene encoding any one of the proteins (A) to (C) (hereinafter also referred to as the gene of the present invention).
  • the present invention also relates to a transformant obtained by introducing a gene encoding any one of the proteins (A) to (C) into a host.
  • the present invention relates to the provision of a method for producing a lipid using an algae-derived acyl-ACP thioesterase, which improves the productivity of a medium-chain fatty acid or a lipid comprising the medium-chain fatty acid.
  • the present invention relates to a novel algae-derived acyl-ACP thioesterase that can be suitably used in the above-described method, and a gene encoding the same.
  • the present invention also relates to the provision of a transformant that promotes the expression of the gene and changes lipid productivity or fatty acid composition.
  • the present inventor has studied a new acyl-ACP thioesterase derived from algae.
  • a novel acyl-ACP thioesterase and an acyl-ACP thioesterase gene encoding the same were found from algae belonging to the genus Nannochloropsis.
  • the present inventors have found that the content of specific fatty acids in the total fatty acid component in the lipid is significantly improved in the transformant.
  • the present invention has been completed based on these findings.
  • a novel acyl-ACP thioesterase, a gene encoding the same, and a transformant into which the gene is introduced can be provided.
  • the production method of the present invention using the transformant is excellent in productivity of medium-chain fatty acids or lipids comprising the same.
  • the production method of the present invention has a carbon number of 8 to 16, preferably 12 to 16, more preferably 12 to 14, more preferably 12 and 14, and more preferably carbon number. Is excellent in productivity of 14 fatty acids or lipids containing these as constituents.
  • the acyl-ACP thioesterase, gene encoding the same, transformant, and production method of the present invention can be suitably used for industrial production of fatty acids or lipids.
  • lipid includes simple lipids, complex lipids and derived lipids, and specifically includes fatty acids, fatty alcohols, hydrocarbons (alkanes, etc.), neutral lipids (triacylglycerols, etc.). , Wax, ceramide, phospholipid, glycolipid, sulfolipid and the like.
  • Cx: y in the notation of fatty acids and acyl groups constituting fatty acids means that the number of carbon atoms is x and the number of double bonds is y.
  • Cx represents a fatty acid or acyl group having x carbon atoms.
  • the identity of a base sequence and an amino acid sequence is calculated by the Lipman-Pearson method (Science, 1985, vol. 227, p. 1435-1441). Specifically, it is calculated by performing an analysis assuming that Unit size to compare (ktup) is 2 using the homology analysis (Search homology) program of genetic information processing software Genetyx-Win.
  • “stringent conditions” include, for example, Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W., et al. Russell., Cold Spring Harbor Laboratory Press].
  • a numerical range expressed using “to” means a range including numerical values described before and after “to” as a lower limit value and an upper limit value.
  • the “medium chain” means that the fatty acid or the fatty acid residue has 8 to 16 carbon atoms.
  • Acyl-ACP thioesterase The protein of the present invention is a protein having an amino acid sequence of at least positions 91 to 348 in the amino acid sequence of SEQ ID NO: 1, and a protein functionally equivalent to the protein.
  • Acyl-ACP (acyl carrier protein) thioesterase is an enzyme involved in the biosynthesis system of fatty acids and derivatives thereof (such as triacylglycerol (triglyceride)).
  • the enzyme contains acyl-ACP (an acyl group that is a fatty acid residue), which is an intermediate in the process of fatty acid biosynthesis, in plastids such as chloroplasts in plants and algae, and in the cytoplasm in bacteria, fungi, and animals.
  • acyl-ACP thioesterase activity refers to an activity of hydrolyzing the thioester bond of acyl-ACP.
  • proteins include the following proteins (A) to (C).
  • a protein comprising the amino acid sequence of positions 91 to 348 of SEQ ID NO: 1.
  • B A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 and having acyl-ACP thioesterase activity.
  • C A protein having the amino acid sequence of the protein (A) or (B) and having acyl-ACP thioesterase activity.
  • SEQ ID NO: 1 is an amino acid sequence of an acyl-ACP thioesterase (hereinafter also abbreviated as NoTE2 ) derived from an algae belonging to the genus Nannochloropsis, Nannochloropsis oculata .
  • NoTE2 acyl-ACP thioesterase
  • the inventor believes that in the amino acid sequence of SEQ ID NO: 1, the region from position 91 to position 348 is important for functioning as an acyl-ACP thioesterase, and is a sufficient region to exhibit acyl-ACP thioesterase activity. I found. That is, a protein consisting of the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 and a protein consisting of an amino acid sequence containing the sequence have acyl-ACP thioesterase activity. Protein (A) has sufficient region for this acyl-ACP thioesterase activity and functions as an acyl-ACP thioesterase.
  • Protein (B) consists of an amino acid sequence having 80% or more identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1, and has acyl-ACP thioesterase activity.
  • an amino acid sequence encoding an enzyme protein does not necessarily indicate enzyme activity unless the sequence of the entire region is conserved, and there are regions that do not affect enzyme activity even if the amino acid sequence changes. It is known to exist. In such a region that is not essential for enzyme activity, the original activity of the enzyme can be maintained even if a mutation such as amino acid deletion, substitution, insertion or addition is introduced.
  • a protein that retains acyl-ACP thioesterase activity and has a partially mutated amino acid sequence can also be used.
  • the identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 is preferably 85% or more, more preferably 90% or more, and more than 95% Is more preferable, 96% or more is more preferable, 97% or more is further more preferable, 98% or more is further more preferable, and 99% or more is further more preferable.
  • the amino acid sequence having 80% or more identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 is one or several in the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1. 1 or more, preferably 20 or less, more preferably 1 or more and 15 or less, still more preferably 1 or more and 10 or less, still more preferably 1 or more and 8 or less, and even more preferably 1 or more 5 or less, more preferably 1 or more and 4 or less, still more preferably 1 or more and 3 or less, and even more preferably 1 or 2 amino acids are deleted, substituted, inserted or added.
  • Examples include sequences.
  • Examples of a method for introducing mutation such as deletion, substitution, insertion, addition, etc. into the amino acid sequence include a method of introducing mutation into the base sequence encoding the amino acid sequence. A method for introducing a mutation into the base sequence will be described later.
  • the protein (C) contains the amino acid sequence of the protein (A) or (B) as part of its amino acid sequence, and exhibits acyl-ACP thioesterase activity.
  • the protein (C) may contain a sequence other than the amino acid sequence of the protein (A) or (B).
  • examples of the sequence other than the amino acid sequence of the protein (A) or (B) include, for example, any amino acid sequence other than positions 91 to 348 of SEQ ID NO: 1,
  • the identity with any amino acid sequence other than positions 91 to 348 of number 1 is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, more preferably 99% or more amino acid sequences, or one or several amino acids in these sequences, preferably 1 or more and 20 or less More preferably, it is 1 or more and 15 or less, more preferably 1 or more and 10 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 5 or less.
  • the protein (C) is also preferably a protein comprising an amino acid sequence in which a signal peptide involved in protein transport or secretion is added to the amino acid sequence of the protein (A) or (B).
  • signal peptide addition include addition of a chloroplast translocation signal peptide to the N-terminus.
  • the protein (C) may be a protein consisting of an amino acid sequence in which the amino acid at the N-terminal side is deleted at any position from 1 to 90 in SEQ ID NO: 1. Furthermore, the following proteins (C1) to (C7) are preferable as the protein (C) from the viewpoint of productivity of specific fatty acids, for example, medium chain fatty acids, specifically, fatty acids having 12 or 14 carbon atoms. .
  • (C1) A protein comprising the amino acid sequence of positions 1 to 348 of SEQ ID NO: 1.
  • C2 A protein comprising the amino acid sequence of positions 61 to 348 of SEQ ID NO: 1.
  • C3 A protein comprising the amino acid sequence of positions 71 to 348 of SEQ ID NO: 1.
  • (C4) A protein comprising the amino acid sequence of positions 74 to 348 of SEQ ID NO: 1.
  • (C5) A protein comprising the amino acid sequence of positions 81 to 348 of SEQ ID NO: 1.
  • (C6) The identity with any one amino acid sequence of the proteins (C1) to (C5) is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, more More preferably 96% or more, still more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more amino acid sequence, and a protein having acyl-ACP thioesterase activity.
  • (C7) One or several, preferably 1 or more and 20 or less, more preferably 1 or more and 15 or less, more preferably 1 or more in any one amino acid sequence of the proteins (C1) to (C5) 10 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 5 or less, more preferably 1 or more and 4 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2 Protein having an acyl-ACP thioesterase activity, comprising an amino acid sequence in which a single amino acid is deleted, substituted, inserted, or added. The present inventors have confirmed that the proteins (C1) to (C5) have acyl-ACP thioesterase activity.
  • a protein has acyl-ACP thioesterase activity means that, for example, a DNA in which an acyl-ACP thioesterase gene is linked downstream of a promoter that functions in a host cell such as E. coli is introduced into a host cell that lacks the fatty acid degradation system. It can be confirmed by culturing under conditions where the introduced acyl-ACP thioesterase gene is expressed, and analyzing changes in the fatty acid composition in the host cell or culture solution using a method such as gas chromatography analysis. .
  • acyl-ACP thioesterase gene linked downstream of a promoter that functions in a host cell such as Escherichia coli into the host cell and culturing the cell under conditions such that the introduced acyl-ACP thioesterase gene is expressed.
  • Various acyls prepared by the method of Yuan et al. (Yuan L, et al., Proc. Natl. Acad. Sci. USA, 1995, vol. 92 (23), p. 10639-10643).
  • -Acyl-ACP thioesterase activity can be measured by carrying out a reaction using ACP as a substrate.
  • a protein derived from a natural product can be obtained by isolation, purification or the like from Nannochloropsis oculata.
  • protein synthesis may be performed by chemical synthesis, or a recombinant protein may be produced by a gene recombination technique.
  • the acyl-ACP thioesterase gene described later can be used.
  • Algae belonging to the genus Nannochloropsis such as Nannochloropsis oculata can also be obtained from a preservation organization such as a private or public laboratory.
  • the acyl-ACP thioesterase gene of the present invention is a gene encoding any one of the proteins (A) to (C).
  • An example of a gene encoding any one of proteins (A) to (C) is a gene having the base sequence shown in SEQ ID NO: 2.
  • the base sequence shown in SEQ ID NO: 2 is an example of the base sequence of the gene encoding acyl-ACP thioesterase derived from Nannochloropsis oculiata, and encodes the amino acid sequence shown in SEQ ID NO: 1.
  • the base sequence from positions 271 to 1044 of SEQ ID NO: 2 encodes the amino acid sequence from positions 91 to 348 of SEQ ID NO: 1. Note that the nucleotide sequence from position 1045 to position 1047 of SEQ ID NO: 2 is a stop codon and does not correspond to an amino acid.
  • a gene consisting of any one of the following DNAs (a) to (c) can be exemplified.
  • the present invention is not limited to these.
  • DNA (b) from the viewpoint of acyl-ACP thioesterase activity, the identity with the nucleotide sequence at positions 271 to 1047 of SEQ ID NO: 2 is preferably 85% or more, more preferably 90% or more, and more than 95% Is more preferable, 96% or more is more preferable, 97% or more is further more preferable, 98% or more is further more preferable, and 99% or more is further more preferable.
  • the nucleotide sequence having 80% or more identity with the nucleotide sequence of positions 271 to 1047 of SEQ ID NO: 2 is one or several in the nucleotide sequence of positions 271 to 1047 of SEQ ID NO: 2.
  • More preferred is a base sequence in which 1 to 4 or less, more preferably 1 to 3 or less, more preferably 1 or 2 bases are deleted, substituted, inserted or added. Examples of methods for introducing mutations such as deletion, substitution, insertion and addition into the base sequence include site-specific mutagenesis.
  • kits such as Site-Directed Mutagenesis System Mutan-SuperExpress Km Kit (Takara Bio), Transformer TM Site-Directed Mutagenesis Kit (Clonetech), KOD-Plus-Mutagenesis Kit (Toyobo) may be used. it can.
  • the target gene after giving a random gene mutation, the target gene can also be obtained by performing enzyme activity evaluation and gene analysis by an appropriate method.
  • DNA (b) DNA that hybridizes with DNA consisting of a base sequence complementary to DNA (a) under stringent conditions and encodes a protein having acyl-ACP thioesterase activity is also preferable.
  • DNA (c) contains the base sequence of DNA (a) or (b) as part of its base sequence, and encodes a protein having acyl-ACP thioesterase activity.
  • the DNA (c) may contain a sequence other than the base sequence of the DNA (a) or (b).
  • sequences other than the base sequence of DNA (a) or (b) in the base sequence of DNA (c) include, for example, any base sequence other than positions 271 to 1047 in SEQ ID NO: 2, SEQ ID NO: 2 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and still more preferably 96%, with any nucleotide sequence other than positions 271 to 1047 Or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more, or any nucleotide sequence other than positions 271 to 1047 in SEQ ID NO: 2 Or several, preferably 1 to 20 or less, more preferably 1 to 15 or less, more preferably 1 to 10 or less, more preferably 1 to 8 or less, more preferably 1 or less.
  • a base sequence encoding a signal peptide involved in protein transport or secretion is also preferable.
  • the signal peptide include those described for the protein (C). These sequences are preferably added to the 5 ′ end side of the base sequence of the DNA (a) or (b).
  • the DNA (c) may be a DNA consisting of a base sequence in which the 5 ′ end is deleted at any position from positions 1 to 270 of SEQ ID NO: 2. Furthermore, from the viewpoint of productivity of specific fatty acids, for example, medium chain fatty acids, specifically, fatty acids having 12 or 14 carbon atoms, the following DNA (c1) to (c7) are preferable as the DNA (c). .
  • DNA consisting of the nucleotide sequence of positions 220 to 1047 of SEQ ID NO: 2.
  • DNA consisting of the nucleotide sequence of positions 241 to 1047 of SEQ ID NO: 2.
  • the identity with any one of the base sequences of DNA (c1) to (c5) is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, more More preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, and encodes a protein having acyl-ACP thioesterase activity DNA.
  • (c7) One or several, preferably 1 or more, 20 or less, more preferably 1 or more and 15 or less, more preferably 1 or more, in any one of the base sequences of DNA (c1) to (c5) 10 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 5 or less, more preferably 1 or more and 4 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2
  • the method for obtaining the acyl-ACP thioesterase gene of the present invention is not particularly limited, and can be obtained by ordinary genetic engineering techniques.
  • a gene can be obtained by artificial synthesis based on the amino acid sequence shown in SEQ ID NO: 1 or the base sequence shown in SEQ ID NO: 2.
  • services such as Eurofin Genomics can be used for artificial gene synthesis.
  • It can also be obtained by cloning from Nannochloropsis oculata, for example, by the method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION [Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press (2001)] be able to.
  • a first aspect of the transformant of the present invention is a transformant in which expression of a gene encoding any one of the proteins (A) to (C) is promoted. It is.
  • the ability to produce lipids particularly the ability to produce medium chain fatty acids having 12 to 16 carbon atoms or lipids comprising them (the production amount and production of lipids comprising medium chain fatty acids or these constituents)
  • the proportion of medium chain fatty acids in the total fatty acids produced and the proportion of lipids comprising the medium chain fatty acids in the total lipid produced as a constituent) are significantly improved.
  • the present invention using the transformant is a specific lipid, particularly a medium chain fatty acid or a lipid comprising this, preferably a fatty acid having 8 to 16 carbon atoms or a lipid comprising this.
  • the fatty acid having 12 or more and 16 or less carbon atoms or a lipid comprising the same more preferably a fatty acid having 12 or more and 14 or less carbon atoms or a lipid comprising the same, more preferably 12 or 14 carbon atoms.
  • acyl-ACP thioesterase to produce fatty acids or lipids can be measured by the method used in the examples.
  • those in which the expression of the gene encoding the target protein is promoted are also referred to as “transformants”, and those in which the expression of the gene encoding the target protein is not promoted are “host” or “ Also referred to as “wild strain”.
  • the method for promoting the expression of the acyl-ACP thioesterase gene can be appropriately selected from conventional methods. For example, a method of introducing the acyl-ACP thioesterase gene into a host, a method of modifying an expression control region (promoter, terminator, etc.) of the gene in a host having the acyl-ACP thioesterase gene on the genome, etc. Can be mentioned.
  • a method for promoting the expression of the gene by introducing the acyl-ACP thioesterase gene into the host will be described.
  • a transformant that can be preferably used in the present invention can be obtained by introducing a gene encoding an acyl-ACP thioesterase into a host by an ordinary genetic engineering method. Specifically, by preparing an expression vector or gene expression cassette capable of expressing a gene encoding an acyl-ACP thioesterase in a host cell, and introducing the gene into a host cell to transform the host cell. Can be made.
  • the host of the transformant is not particularly limited and can be appropriately selected from those usually used.
  • microorganisms including algae and microalgae
  • plants, or animals can be used.
  • the host is preferably a microorganism or a plant, and more preferably a microorganism.
  • the microorganism may be either prokaryotic, eukaryotic, Escherichia (Escherichia) microorganism belonging to the genus microorganism belonging to Bacillus (Bacillus) genus Synechocystis (Synechocystis) microorganism of the genus, Synechococcus (Synechococcus) microorganisms of the genus Or eukaryotic microorganisms such as yeast and filamentous fungi can be used. Among them, from the viewpoint of the lipid productivity, E.
  • Escherichia coli is a microorganism belonging to Escherichia
  • Bacillus subtilis Bacillus subtilis
  • Bacillus subtilis Bacillus subtilis
  • red yeast is a microorganism belonging to yeast (Rhodosporidium toruloides), Or Mortierella sp.
  • which is a microorganism belonging to filamentous fungi is preferable, and Escherichia coli is more preferable.
  • algae belonging to the genus Chlamydomonas from the viewpoint of establishing genetic recombination techniques, algae belonging to the genus Chlamydomonas , algae belonging to the genus Chlorella , algae belonging to the genus Phaeodactylum , Or algae belonging to the genus Nannochloropsis are preferable, and algae belonging to the genus Nannochloropsis are more preferable.
  • Nannochloropsis gaditana Specific algae belonging to the genus Nannochloropsis are Nannochloropsis gaditana , Nannochloropsis salina , Nannochloropsis oceanica , Nannochloropsis oceanica , Nannochloropsis oceanina , Examples thereof include Nannochloropsis atomus , Nannochloropsis maculata , Nannochloropsis granulata , Nannochloropsis sp., And the like. Among these, from the viewpoint of lipid productivity, Nannochloropsis oculata or Nannochloropsis gaditana is preferable, and Nannochloropsis oculata is more preferable.
  • the plant body is preferably Arabidopsis thaliana , rapeseed, coconut palm, palm, coffea, or jatropha, more preferably Arabidopsis , from the viewpoint of high lipid content in the seed.
  • a gene encoding acyl-ACP thioesterase can be introduced into a host, and the gene can be expressed in the host cell.
  • Any vector may be used.
  • a vector having an expression regulatory region such as a promoter or terminator according to the type of host to be introduced, and a vector having a replication origin or a selection marker can be used.
  • it may be a vector that autonomously grows and replicates outside the chromosome, such as a plasmid, or a vector that is integrated into the chromosome.
  • pBluescript II SK ( ⁇ ) or pMW218 / 219 is preferably used.
  • algae for example, pUC19 (manufactured by Takara Bio Inc.), P66 (Chlamydomonas Center), P-322 (Chlamydomonas Center), pPha-T1 (Yangmin Gong, et al., Journal of Basic Microbiology, 2011) , Vol.51, p.666-672), or pJET1 (manufactured by Cosmo Bio).
  • a host when the host is an algae belonging to the genus Nannochloropsis, pUC19, pPha-T1, or pJET1 is preferably used.
  • a host When the host is an algae belonging to the genus Nannochloropsis, Oliver Kilian, et al., Proceedings of the National Academy of Sciences of the United States of America, 2011, vol.
  • a host can also be transformed with a DNA fragment (gene expression cassette) comprising the gene, promoter and terminator of the present invention.
  • the DNA fragment include a DNA fragment amplified by PCR and a restriction enzyme-cleaved DNA fragment.
  • a pRI vector manufactured by Takara Bio Inc.
  • a pBI vector manufactured by Clontech
  • an IN3 vector manufactured by Implanta Innovations
  • the host is Arabidopsis thaliana
  • pRI vectors or pBI vectors are preferably used.
  • the type of promoter or terminator that regulates the expression of the gene encoding the target protein incorporated above can also be appropriately selected according to the type of host used.
  • a promoter that can be preferably used in the present invention for example, by adding lac promoter, trp promoter, tac promoter, trc promoter, T7 promoter, SpoVG promoter, isopropyl ⁇ -D-1-thiogalactopyranoside (IPTG) Promoters related to inducible derivatives, Rubisco operon (rbc), PSI reaction center protein (psaAB), PSII D1 protein (psbA), cauliflower mosaic virus 35SRNA promoter, housekeeping gene promoter (eg tubulin promoter, actin promoter, ubiquitin) Promoter, rapeseed-derived Napin gene promoter, plant-derived Rubisco promoter, violaxanthin / chlorophyll a-binding protein VCP1 gene promoter from the genus Nannochloropsis (V
  • LDSP lipid droplet surface protein
  • the type of selectable marker for confirming that the gene encoding the target protein has been incorporated can be appropriately selected according to the type of host used.
  • Selectable markers that can be preferably used in the present invention include ampicillin resistance gene, chloramphenicol resistance gene, erythromycin resistance gene, neomycin resistance gene, kanamycin resistance gene, spectinomycin resistance gene, tetracycline resistance gene, blasticidin S Examples include drug resistance genes such as resistance genes, bialaphos resistance genes, zeocin resistance genes, paromomycin resistance genes, or hygromycin resistance genes. Furthermore, it is also possible to use a gene defect associated with auxotrophy as a selection marker gene.
  • Introduction of a gene encoding a target protein into the vector can be performed by a usual technique such as restriction enzyme treatment or ligation.
  • sequences useful for translation of the gene for example, sequences corresponding to a start codon and a stop codon can be appropriately supplemented.
  • the transformation method is not particularly limited as long as it is a method capable of introducing a target gene into a host. For example, a method using calcium ions, a general competent cell transformation method (J. Bacterial. 93, 1925 (1967)), a protoplast transformation method (Mol. Gen. Genet.
  • ⁇ Selection of transformant introduced with target gene fragment> can be performed by using a selection marker or the like.
  • the drug resistance gene acquired by the transformant as a result of introducing a vector-derived drug resistance gene into the host cell together with the target DNA fragment at the time of transformation can be used as an indicator.
  • the introduction of the target DNA fragment can also be confirmed by PCR method using a genome as a template.
  • “Expression regulatory region” refers to a promoter or terminator, and these sequences are generally involved in regulating the expression level (transcription level, translation level) of adjacent genes.
  • “Expression regulatory region” refers to a promoter or terminator, and these sequences are generally involved in regulating the expression level (transcription level, translation level) of adjacent genes.
  • Examples of the method for modifying the expression regulatory region include promoter replacement.
  • the acyl-ACP thioesterase gene promoter (hereinafter also referred to as “acyl-ACP thioesterase promoter”) is replaced with a promoter having a higher transcriptional activity, whereby the acyl- Expression of the ACP thioesterase gene can be promoted.
  • an acyl-ACP thioesterase is directly under the DNA sequence consisting of the nucleotide sequence shown in SEQ ID NO: 37.
  • the gene is present, and a promoter region is present in the DNA sequence consisting of the base sequence shown in SEQ ID NO: 37.
  • the promoter used for replacement of the acyl-ACP thioesterase promoter is not particularly limited, and is appropriately selected from those having higher transcription activity than the acyl-ACP thioesterase promoter and suitable for the production of medium-chain fatty acids or lipids comprising them. You can choose.
  • the promoter of the LDSP gene can be preferably used. From the viewpoint of improving the productivity of medium-chain fatty acids or lipids containing these as constituents, the promoter of violaxanthin / chlorophyll a-binding protein gene and the promoter of LDSP gene are more preferable.
  • the above-described promoter modification can be performed according to a conventional method such as homologous recombination. Specifically, a linear DNA fragment containing upstream and downstream regions of the target promoter and containing another promoter instead of the target promoter is constructed and incorporated into the host cell, and the target promoter of the host genome Two homologous recombination occurs at the upstream and downstream side of. As a result, the target promoter on the genome is replaced with another promoter fragment, and the promoter can be modified.
  • a method for modifying a target promoter by homologous recombination is described in, for example, Besher et al., Methods in molecular biology, 1995, vol. 47, p. Reference can be made to documents such as 291-302.
  • a second aspect of the transformant of the present invention is a transformant in which the gene is deleted, mutated or expression-suppressed in a host having the acyl-ACP thioesterase gene of the present invention. is there.
  • the transformant (hereinafter also referred to as “transformant of the second aspect”) is a deletion, mutation, or mutation of a gene encoding any one of the proteins (A) to (C) in the host. It is obtained by suppressing expression.
  • the host of the transformant of the second aspect is not particularly limited as long as it has the acyl-ACP thioesterase gene of the present invention, and microorganisms, plants, or animals can be used.
  • microorganisms are preferable, and Escherichia coli or microalgae are more preferable.
  • the microalgae are preferably algae belonging to the genus Nannochloropsis from the viewpoint of lipid productivity.
  • Nannochloropsis oculata examples include Nannochloropsis oculata, Nannochloropsis gaditana, Nannochloropsis salina, Nannochloropsis oceanica, Nannochloropsis Atoms, Nannochloropsis maculata, Nanno Chloropsis granulata or Nannochloropsis sp.
  • Nannochloropsis oculata or Nannochloropsis gaditana is preferable, and Nannochloropsis oculata is more preferable.
  • a part or all of the target gene is removed from the genome or replaced with another gene.
  • This method can be carried out by inserting a DNA fragment of this gene, or by mutating the active site, substrate binding site or transcription / translation initiation region of the gene.
  • the above-described deletion / mutation / expression suppression method can be performed using, for example, homologous recombination.
  • a linear DNA fragment that contains upstream and downstream regions of the target gene but does not contain the target gene is constructed by a method such as PCR, and is incorporated into the host cell to upstream of the target gene in the host genome.
  • the target gene on the genome can be deleted or replaced with another gene fragment by causing homologous recombination twice on the side and downstream.
  • a target gene into which a mutation such as base substitution or base insertion has been introduced is constructed by a method such as PCR, and this is incorporated into a host cell, and twice at two locations outside the mutation site in the target gene of the host genome.
  • a circular recombinant plasmid obtained by cloning a DNA fragment containing a part of the target gene into an appropriate plasmid vector is incorporated into the host cell, and the host genome is obtained by homologous recombination in a part of the target gene.
  • the target gene above can be disrupted to reduce or eliminate its function.
  • target gene deletion / mutation / expression suppression methods by homologous recombination are described in, for example, Besher et al., Methods in molecular biology, 1995, vol. 47, p.291-302.
  • the host is an algae belonging to the genus Nannochloropsis
  • homology with reference to documents such as Oliver Kilian, et al., Proceedings of the National Academy of Sciences of the United States of America, 2011, 108 (52)
  • Specific genes in the genome can be deleted or destroyed by recombinant methods.
  • genomic DNA is extracted from the transformants and PCR is performed on the region containing the target gene site, or a DNA probe that binds to the target gene region is used.
  • the Southern blotting method can be used.
  • the fatty acid composition of the produced lipid is considered to change from the original composition of the host. That is, the transformant can produce a lipid having a modified fatty acid composition in the lipid.
  • the transformant of the first aspect of the present invention has improved productivity of medium chain fatty acids or lipids comprising this as a constituent, compared to the host. Therefore, if the transformant of the present invention is cultured under appropriate conditions, and then the medium chain fatty acid or the lipid comprising this as a constituent component is recovered from the obtained culture, the medium chain fatty acid or the lipid comprising this constituent is obtained. Can be manufactured efficiently.
  • the method for producing a lipid of the present invention comprises a step of culturing a transformant introduced with a gene encoding acyl-ACP thioesterase under appropriate conditions to obtain a culture from the viewpoint of improving lipid productivity, and It is preferable to include a step of collecting lipid from the obtained culture.
  • culturing a transformant means culturing and growing a microorganism, algae, a plant, an animal, and cells and tissues thereof, and includes cultivating the plant in soil or the like.
  • the “culture” includes, in addition to the culture solution, the transformant itself after culturing and the like.
  • Culture conditions can be appropriately selected depending on the host of the transformant, and culture conditions generally used for the host can be used. From the viewpoint of lipid production efficiency, for example, glycerol, acetic acid, malonic acid or the like may be added to the medium as a substrate of acyl-ACP thioesterase or a precursor involved in the fatty acid biosynthesis system.
  • a transformant using Escherichia coli as a host culturing in an LB medium or Overnight Express Instant TB Medium (Novagen) at 30 to 37 ° C. for 0.5 to 1 day can be mentioned.
  • a transformant using Arabidopsis as a host it is cultivated for 1 to 2 months in soil under a temperature condition of 20 to 25 ° C. and under a light condition such as continuous irradiation with white light or a light period of 16 hours and a dark period of 8 hours. Can be mentioned.
  • the culture medium may be based on natural seawater or artificial seawater, or a commercially available culture medium may be used.
  • the medium include f / 2 medium, ESM medium, Daigo IMK medium, L1 medium, and MNK medium.
  • f / 2 medium, ESM medium, or Daigo IMK medium is preferable, f / 2 medium or Daigo IMK medium is more preferable, and f / 2 medium is further included. preferable.
  • nitrogen sources, phosphorus sources, metal salts, vitamins, trace metals, and the like can be appropriately added to the medium.
  • the amount of algae inoculated on the medium is not particularly limited, but is preferably 1 to 50% (vol / vol), more preferably 1 to 10% (vol / vol) per medium from the viewpoint of growth.
  • the culture temperature is not particularly limited as long as it does not adversely affect the growth of algae, but it is usually in the range of 5 to 40 ° C. From the viewpoint of promoting the growth of algae, improving the productivity of fatty acids, and reducing the production cost, the temperature is preferably 10 to 35 ° C, more preferably 15 to 30 ° C.
  • the light irradiation may be performed under conditions that allow photosynthesis, and may be artificial light or sunlight.
  • the illuminance during light irradiation is preferably in the range of 100 to 50000 lux, more preferably in the range of 300 to 10000 lux, and still more preferably in the range of 1000 to 6000 lux, from the viewpoint of promoting the growth of algae and improving the productivity of fatty acids. It is. Further, the light irradiation interval is not particularly limited, but from the same viewpoint as described above, it is preferably performed in a light / dark cycle, and the light period in 24 hours is preferably 8 to 24 hours, more preferably 10 to 18 hours, More preferably, it is 12 hours.
  • the concentration of carbon dioxide in the gas is not particularly limited, but is preferably 0.03 (similar to atmospheric conditions) to 10%, more preferably 0.05 to 5%, from the viewpoint of promoting growth and improving the productivity of fatty acids. More preferably, it is 0.1 to 3%, and still more preferably 0.3 to 1%.
  • the concentration of the carbonate is not particularly limited. For example, when sodium bicarbonate is used, it is preferably 0.01 to 5% by mass, more preferably 0.05 to 2% by mass, from the viewpoint of promoting growth and improving the productivity of fatty acids.
  • the content is 0.1 to 1% by mass.
  • the culture time is not particularly limited, and may be performed for a long period of time (for example, about 150 days) so that algal bodies that accumulate lipids at a high concentration can grow at a high concentration.
  • the culture period is preferably 3 to 90 days, more preferably 3 to 30 days, and even more preferably 7 to 30 days.
  • culture cultivation may be any of aeration stirring culture, shaking culture, or stationary culture, and aeration stirring culture is preferable from the viewpoint of improving aeration.
  • a method for collecting lipid produced in the transformant a method usually used for isolating lipid components in a living body, for example, filtration, centrifugation, cell culture from the aforementioned culture or transformant
  • a lipid component is isolated and recovered by crushing, gel filtration chromatography, ion exchange chromatography, chloroform / methanol extraction method, hexane extraction method or ethanol extraction method.
  • oil can be recovered from the culture or transformant by pressing or extraction, and then subjected to general purification such as degumming, deoxidation, decolorization, dewaxing, and deodorization to obtain lipids. .
  • a fatty acid can be obtained by hydrolyzing the isolated lipid.
  • the method for isolating the fatty acid from the lipid component include a method of treating at a high temperature of about 70 ° C. in an alkaline solution, a method of treating with lipase, a method of decomposing using high-pressure hot water, and the like.
  • the acyl-ACP thioesterase of the present invention has high specificity for medium chain acyl-ACP, further C12-C16 acyl-ACP, especially C12 and C14 acyl-ACP.
  • the production method of the present invention using the transformant is suitable for lipid production, particularly medium chain fatty acids, preferably fatty acids having 8 to 16 carbon atoms, more preferably fatty acids having 12 to 16 carbon atoms, more preferably carbon numbers. It can be suitably used for the production of fatty acids having 12 to 14 fatty acids, more preferably fatty acids having 12 and 14 carbon atoms, more preferably fatty acids having 14 carbon atoms, or lipids composed thereof.
  • the lipid produced in the production method of the present invention preferably contains a fatty acid or a fatty acid compound, and more preferably contains a fatty acid or a fatty acid ester compound thereof, from the viewpoint of its availability.
  • the lipid produced in the production method of the present invention is preferably a fatty acid having 8 to 16 carbon atoms or a fatty acid ester compound thereof, more preferably a fatty acid having 12 to 16 carbon atoms or a fatty acid ester compound thereof. More preferably, the fatty acid having 12 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a fatty acid having 12 and 14 carbon atoms or a fatty acid ester compound thereof, more preferably a fatty acid having 14 carbon atoms or a fatty acid ester compound thereof. including.
  • the fatty acid or fatty acid ester compound contained in the lipid is preferably a fatty acid having 8 to 16 carbon atoms or a fatty acid ester thereof, more preferably a fatty acid ester having 12 to 16 carbon atoms or a fatty acid ester thereof, from the viewpoint of availability to a surfactant or the like.
  • a fatty acid having 12 to 14 carbon atoms or a fatty acid ester compound thereof is more preferable
  • a fatty acid having 12 or 14 carbon atoms or a fatty acid ester compound thereof is more preferable
  • a fatty acid having 14 carbon atoms or a fatty acid ester compound thereof is more preferable.
  • the fatty acid ester compound is preferably a simple lipid or a complex lipid, more preferably a simple lipid, and even more preferably triacylglycerol.
  • Fatty acids and lipids obtained by the production method of the present invention and transformants are used as food, as emulsifiers for cosmetics, detergents such as soaps and detergents, fiber treatment agents, hair rinse agents, or bactericides and preservatives. Can be used.
  • the present invention further discloses the following methods, transformants, proteins, and genes.
  • a method for producing lipid comprising culturing a transformant into which a gene encoding any one of the following proteins (A) to (C) is introduced into a host, and collecting the lipid from the culture.
  • a protein comprising the amino acid sequence of positions 91 to 348 of SEQ ID NO: 1.
  • B A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 and having acyl-ACP thioesterase activity.
  • C A protein having the amino acid sequence of the protein (A) or (B) and having acyl-ACP thioesterase activity.
  • ⁇ 2> Fatty acid produced in the cells of the transformant or lipid comprising this as a constituent, comprising the step of introducing a gene encoding any one of the proteins (A) to (C) into the host A way to increase productivity.
  • lipid is a medium-chain fatty acid or a lipid containing this as a constituent.
  • a medium-chain fatty acid produced in a cell of the transformant comprising a step of introducing a gene encoding any one of the proteins (A) to (C) into the host to obtain a transformant,
  • a method for modifying a lipid composition which improves the productivity of lipids comprising this as a constituent component, and modifies the total fatty acids produced or the composition of fatty acids or lipids in the total lipids.
  • ⁇ 5> A method for producing lipid, comprising culturing a transformant in which expression of a gene encoding any one of the proteins (A) to (C) is promoted, and collecting lipid from the culture.
  • ⁇ 6> A fatty acid produced in a cell of a transformant or a lipid comprising the same, comprising a step of promoting the expression of a gene encoding any one of the proteins (A) to (C). A way to increase productivity.
  • ⁇ 7> The method according to ⁇ 6>, wherein the lipid is a medium-chain fatty acid or a lipid comprising this as a constituent.
  • ⁇ 8> Medium chain fatty acids produced in cells of transformants by promoting the expression of a gene encoding any one of the proteins (A) to (C) or lipids comprising the same
  • a method for modifying the composition of lipids which improves productivity and modifies the composition of fatty acids or lipids in total fatty acids or total lipids produced.
  • ⁇ 9> The method according to any one of ⁇ 5> to ⁇ 9>, wherein a gene encoding any one of the proteins (A) to (C) is introduced into a host to promote expression of the gene. Method.
  • the identity with the amino acid sequence at positions 91 to 348 of SEQ ID NO: 1 is 85% or more, preferably 90% or more, more preferably 95% or more, more preferably 96% or more.
  • the protein (B) has one or several, preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 15 amino acid sequences of SEQ ID NO: 1.
  • ⁇ 12> The protein according to any one of the above ⁇ 1> to ⁇ 9>, wherein the protein (C) is composed of an amino acid sequence in which the amino acid at the N-terminal side is deleted at any position from 1 to 90 in SEQ ID NO: 1.
  • the method described. ⁇ 13> The method according to any one of ⁇ 1> to ⁇ 9>, wherein the protein (C) is any one of the following proteins (C1) to (C7).
  • C1 A protein comprising the amino acid sequence of positions 1 to 348 of SEQ ID NO: 1.
  • C2 A protein comprising the amino acid sequence of positions 61 to 348 of SEQ ID NO: 1.
  • C3 A protein comprising the amino acid sequence of positions 71 to 348 of SEQ ID NO: 1.
  • (C4) A protein comprising the amino acid sequence of positions 74 to 348 of SEQ ID NO: 1.
  • (C5) A protein comprising the amino acid sequence of positions 81 to 348 of SEQ ID NO: 1.
  • (C6) The identity with any one amino acid sequence of the proteins (C1) to (C5) is 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, more preferably Is a protein having an amino acid sequence of 96% or more, more preferably 97% or more, more preferably 98% or more, and still more preferably 99% or more, and having acyl-ACP thioesterase activity.
  • (C7) One or several, preferably 1 or more and 20 or less, more preferably 1 or more and 15 or less, more preferably 1 or more in any one amino acid sequence of the proteins (C1) to (C5) 10 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 5 or less, more preferably 1 or more and 4 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2 Protein having an acyl-ACP thioesterase activity, comprising an amino acid sequence in which a single amino acid is deleted, substituted, inserted, or added.
  • ⁇ 14> The above ⁇ 1> to ⁇ 13, wherein the gene encoding any one of the proteins (A) to (C) is a gene consisting of any one of the following DNA (a) to (c): The method of any one of>.
  • DNA encoding a protein having the base sequence of DNA (a) or (b) and having acyl-ACP thioesterase activity (c) DNA encoding a protein having the base sequence of DNA (a) or (b) and having acyl-ACP thioesterase activity.
  • the DNA (b) has one or more, preferably 1 to 20, more preferably 1 to 15, more preferably 1 or more nucleotide sequences in the DNA (a).
  • DNA (c) comprises a base sequence deleted at the 5′-terminal side at any position from positions 1 to 270 of SEQ ID NO: 2.
  • DNA (c) is any one of the following DNA (c1) to (c7).
  • (c1) DNA consisting of the nucleotide sequence of positions 1 to 1047 of SEQ ID NO: 2.
  • c2 DNA consisting of the nucleotide sequence of positions 181 to 1047 of SEQ ID NO: 2.
  • c3 DNA consisting of the nucleotide sequence of positions 211 to 1047 of SEQ ID NO: 2.
  • DNA consisting of the nucleotide sequence of positions 220 to 1047 of SEQ ID NO: 2.
  • DNA consisting of the nucleotide sequence of positions 241 to 1047 of SEQ ID NO: 2.
  • the identity with any one of the base sequences of DNA (c1) to (c5) is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, more More preferably 96% or more, still more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, and encodes a protein having acyl-ACP thioesterase activity DNA.
  • (c7) One or several, preferably 1 or more, 20 or less, more preferably 1 or more and 15 or less, more preferably 1 or more, in any one of the base sequences of DNA (c1) to (c5) 10 or less, more preferably 1 or more and 8 or less, more preferably 1 or more and 5 or less, more preferably 1 or more and 4 or less, more preferably 1 or more and 3 or less, more preferably 1 or 2
  • ⁇ 18> The method according to any one of ⁇ 1> to ⁇ 17>, wherein the host of the transformant is a microorganism. ⁇ 19> The method according to ⁇ 18>, wherein the microorganism is Escherichia coli. ⁇ 20> The method according to ⁇ 18>, wherein the microorganism is a microalgae. ⁇ 21> The method according to ⁇ 20>, wherein the microalgae are algae belonging to the genus Nannochloropsis, preferably Nannochloropsis oculata.
  • the lipid is a medium-chain fatty acid or a fatty acid ester compound thereof, preferably a fatty acid having 8 to 16 carbon atoms or a fatty acid ester compound thereof, more preferably a fatty acid having 12 to 16 carbon atoms or a fatty acid ester compound thereof, more preferably.
  • a fatty acid having 12 to 14 carbon atoms or a fatty acid ester compound thereof more preferably a fatty acid having 12 and 14 carbon atoms or a fatty acid ester compound thereof, still more preferably a fatty acid having 14 carbon atoms or a fatty acid ester compound thereof,
  • the method according to any one of ⁇ 1> to ⁇ 21>.
  • ⁇ 24> A gene encoding the protein according to ⁇ 23>.
  • ⁇ 25> A gene comprising any one of DNA (a) to (c) defined in any one of ⁇ 1> to ⁇ 22>.
  • ⁇ 26> A recombinant vector containing the gene according to ⁇ 24> or ⁇ 25>.
  • ⁇ 27> A transformant obtained by introducing the gene according to ⁇ 24> or ⁇ 25> or the recombinant vector according to ⁇ 26> into a host.
  • ⁇ 28> A method for producing a transformant, wherein the gene according to ⁇ 24> or ⁇ 25> or the recombinant vector according to ⁇ 26> is introduced into a host.
  • ⁇ 29> A transformant that promotes the expression of the gene according to ⁇ 24> or ⁇ 25>.
  • ⁇ 30> The transformant according to any one of ⁇ 27> to ⁇ 29> or a method for producing the transformant, wherein a host of the transformant is a microorganism.
  • ⁇ 31> The transformant according to ⁇ 30> or the method for producing the same, wherein the microorganism is Escherichia coli.
  • ⁇ 32> The transformant according to ⁇ 30> or a method for producing the same, wherein the microorganism is a microalgae.
  • ⁇ 33> The transformant according to ⁇ 32> or the method for producing the same, wherein the microalgae is an algae belonging to the genus Nannochloropsis, preferably Nannochloropsis oculata.
  • the lipid is a medium chain fatty acid or a fatty acid ester compound thereof, preferably a fatty acid having 8 to 16 carbon atoms or a fatty acid ester thereof, more preferably a fatty acid having 12 to 16 carbon atoms or a fatty acid ester compound thereof, More preferably a fatty acid having 12 to 14 carbon atoms or a fatty acid ester compound thereof, more preferably a fatty acid having 12 and 14 carbon atoms or a fatty acid ester compound thereof, still more preferably a fatty acid having 14 carbon atoms or a fatty acid ester compound thereof,
  • the use according to ⁇ 34> above, comprising ⁇ 36> A method for modifying a fatty acid composition in a lipid, comprising a step
  • Example 1 Acquisition of acyl-ACP thioesterase gene, transformation of E. coli, and production of lipid by transformant
  • Acquisition of acyl-ACP thioesterase gene derived from Nannochloropsis oculata Nannochloropsis oculata NIES2145 strain Total RNA from National Institute for Environmental Studies (NIES) is extracted, and cDNA is obtained by reverse transcription using SuperScript TM III First-Strand Synthesis SuperMix for qRT-PCR (manufactured by Invitrogen). It was.
  • cDNA Using this cDNA as a template, a gene fragment consisting of the base sequence shown in SEQ ID NO: 2 was obtained by PCR using the primer pair of primer No. 3 and primer No. 4 shown in Table 1.
  • NoTE2 gene this gene is referred to as NoTE2 gene, and the protein encoded by the gene is referred to as NoTE2 (SEQ ID NO: 1).
  • pBluescriptII SK (-) was amplified by PCR using the plasmid vector pBluescriptII SK (-) (manufactured by Stratagene) as a template and the primer pair of primer number 5 and primer number 6 shown in Table 1, and the restriction enzyme Dpn I
  • the mold was digested by treatment (manufactured by Toyobo Co., Ltd.). These two fragments were purified using High Pure PCR Product Purification Kit (Roche Applied Science) and then fused using In-Fusion HD Cloning Kit (Clontech).
  • Transformation into a competent cell manufactured by Takara Bio Inc.
  • plasmid extraction and confirmation of the base sequence of the cloned fragment were performed to prepare a plasmid in which the full length of the NoTE2 gene was cloned.
  • NoTE2 gene expression plasmid In the amino acid sequence of NoTE2 shown in SEQ ID NO: 1, the vicinity of N-terminal positions 1 to 73 was estimated to be a chloroplast translocation signal sequence. Therefore, a plurality of NoTE2 gene expression plasmids were constructed in which the N-terminal region containing the putative chloroplast transition signal sequence was deleted at various lengths. Using the plasmid as a template, PCR was performed using a primer pair of any one of the primer numbers 7 to 12 shown in Table 1 and the primer number 5 primer, and the resulting gene fragment was obtained in the same manner as described above.
  • plasmids NoTE2_61, NoTE2_71, NoTE2_74, NoTE2_81, NoTE2_91, and NoTE2_101 for NoTE2 gene expression were constructed.
  • the plasmid NoTE2_61 was constructed so as to remove the amino acid sequence from the 1st to 60th positions on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1, and the NoTE2 gene was converted into the amino acid sequence from positions 61 to 348 of SEQ ID NO: 1. It has the corresponding nucleotide sequence and nucleotide sequence from position 181 to position 1047 of SEQ ID NO: 2 corresponding to the stop codon.
  • the plasmid NoTE2_71, the plasmid NoTE2_74, the plasmid NoTE2_81, the plasmid NoTE2_91, and the plasmid NoTE2_101 are 1 to 70 positions, 1 to 73 positions, 1 to 80 positions, and 1 to 90 positions on the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1. Or an amino acid sequence at positions 1 to 100 was removed.
  • the amino acid sequence at positions 1 to 29 on the N-terminal side of the LacZ protein derived from the plasmid vector pBluescriptII SK (-) is upstream of the site from which the N-terminal side of the amino acid sequence shown in SEQ ID NO: 1 has been removed. It was constructed to express the fused protein.
  • the obtained colonies were inoculated into 2 mL of Overnight Express Instant TB Medium (Novagen) (containing 50 ⁇ g / mL of ampicillin sodium), and cultured with shaking at 160 ° C. (160 rpm). After 24 hours of culture, the lipid components contained in the culture solution were analyzed by the following method. As a negative control, the same experiment was performed for E. coli K27 strain transformed with the plasmid vector pBluescriptII SK (-).
  • Nitrogen gas was blown onto the resulting chloroform layer to dry it, 0.7 mL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was kept constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of a methanol solution of 14% boron trifluoride (manufactured by SIGMA) was added and the temperature was kept constant at 80 ° C. for 10 minutes. Thereafter, 0.5 mL each of hexane and saturated saline was added and stirred vigorously. After standing at room temperature for 10 minutes or longer, the upper hexane layer was recovered to obtain a fatty acid methyl ester.
  • the fatty acid methyl ester was identified by subjecting the sample to gas chromatograph mass spectrometry analysis under the same conditions.
  • the amount of methyl ester of each fatty acid was quantified from the peak area of the waveform data obtained by gas chromatography analysis.
  • Each peak area was compared with the peak area of 7-pentadecanone, which is an internal standard, to correct between samples, and the amount of each fatty acid per liter of culture solution was calculated.
  • the sum total of each fatty acid amount was made into the total fatty acid amount, and the ratio of each fatty acid amount which occupies for the total fatty acid amount was computed.
  • Table 3 The results are shown in Table 3.
  • TFA represents the total fatty acid content
  • Fatty Acid Composition (% TFA)” represents the ratio (weight percent) of each fatty acid to the total fatty acid.
  • Cx: y represents a fatty acid having x carbon atoms and y double bonds
  • C17: 0 ⁇ ” and “C19: 0 ⁇ ” are cis-9,10-methylenehexadecanoic acid, respectively. (Cis-9,10-Methylen-hexadecanoic acid) and cis-11,12-methyleneoctadecanoic acid.
  • the proportions of C12: 0 fatty acid, C14: 1 fatty acid, C14: 0 fatty acid, and C16: 1 fatty acid in the fatty acid increased greatly.
  • the ratio of C14 fatty acids was significantly increased.
  • the total fatty acid amount (TFA) was also increased in these NoTE2 gene expression plasmid-introduced strains. From these results, it was confirmed that the protein encoded by the gene introduced into plasmid NoTE2_61, NoTE2_71, NoTE2_74, NoTE2_81, or NoTE2_91 has acyl-ACP thioesterase activity. In addition, since these proteins markedly increased the ratio and production amount of C12 fatty acid and C14 fatty acid, they are considered to be acyl-ACP thioesterases having high specificity for C12 fatty acid and C14 fatty acid, particularly C14 fatty acid. .
  • the fatty acid composition hardly changed and the total fatty acid amount did not increase as compared with the negative control. From this, it is considered that the protein encoded by the gene introduced into the plasmid NoTE2_101 has almost no acyl-ACP thioesterase activity. From the above results, it is recognized that a protein having at least the 91st to 348th regions in the amino acid sequence represented by SEQ ID NO: 1 exhibits acyl-ACP thioesterase activity.
  • Example 2 Transformation of Nannochloropsis oculata with an acyl-ACP thioesterase gene derived from Nannochloropsis oculata, and production of lipid by the transformant (1) Construction of plasmid for expression of zeocin resistance gene Zeocin resistance gene (SEQ ID NO: SEQ ID NO: 13) and a tubulin promoter sequence (SEQ ID NO: 14) derived from the Nannochloropsis gaditana CCMP526 strain described in the literature (Randor Radakovits, et al., Nature Communications, DOI: 10.1038 / ncomms1688, 2012).
  • PCR was performed using the primer pair of primer number 15 and primer number 16 and the primer pair of primer number 17 and primer number 18 shown in Table 2, and the zeocin resistance gene and the tubulin promoter sequence were obtained. Each was amplified. Further, PCR was performed using the genome of Nannochloropsis oculata NIES2145 strain as a template and the primer pair of primer number 19 and primer number 20 shown in Table 2 to amplify the heat shock protein terminator sequence (SEQ ID NO: 21).
  • PCR was carried out using the primer pair of primer number 22 and primer number 23 shown in Table 2 to amplify the plasmid vector pUC19.
  • PCR was performed using the above-mentioned zeocin resistance gene expression plasmid as a template and the primer pair of primer number 32 and primer number 23 shown in Table 2, and a zeocin resistance gene expression cassette (tubulin promoter sequence, zeocin resistance gene, heat The fragment consisting of the shock protein terminator sequence) and the pUC19 sequence was amplified.
  • NoTE2 gene expression plasmid (NoTE2-Nanno).
  • the plasmid consists of an LDSP promoter sequence, a NoTE2 gene, a VCP1 terminator sequence, a tubulin promoter sequence, a zeocin resistance gene, and a heat shock protein terminator sequence in that order, and a pUC19 vector sequence.
  • a gene fragment was obtained by PCR using the NoTE2 gene expression plasmid (NoTE2-Nanno) as a template and the primer pair of primer number 33 and primer number 27 shown in Table 2. Further, PCR using the primer pair of primer number 34 and primer number 35 shown in Table 2 was performed using the cDNA of Nannochloropsis oculata strain NIES2145 as a template, and a VCP1 chloroplast transition signal (SEQ ID NO: 36) was obtained. . These fragments were fused in the same manner as described above to construct a NoTE2 gene Nannochloropsis expression plasmid (NoTE2_74-Nanno).
  • This plasmid is a NoTE2 gene (hereinafter referred to as “NoTE2_74 gene”) in which the LDSP promoter sequence and VCP1 chloroplast translocation signal are linked to the 5 ′ end of the nucleotide sequence encoding positions 74 to 348 of the amino acid sequence shown in SEQ ID NO: 1. "), A VCP1 terminator sequence, a tubulin promoter sequence, a zeocin resistance gene, a heat shock protein terminator sequence in that order, and a pUC19 vector sequence.
  • NoTE2 gene expression cassette into Nannochloropsis oculata Primers No. 20 and No. 26 shown in Table 2 using the aforementioned NoTE2 gene expression plasmids (NoTE2-Nanno and NoTE2_74-Nanno) as templates, respectively.
  • PCR using the pair was performed to amplify the NoTE2 gene expression cassette (DNA fragment consisting of LDSP promoter sequence, NoTE2 gene or NoTE2_74 gene, VCP1 terminator sequence, tubulin promoter sequence, zeocin resistance gene, heat shock protein terminator sequence).
  • the amplified DNA fragment was purified using High Pure PCR Product Purification Kit (Roche Applied Science). Note that sterilized water was used for elution during purification, not the elution buffer included in the kit.
  • Nannochloropsis oculata strain NIES2145 were washed with a 384 mM sorbitol solution to completely remove salts, and used as host cells for transformation.
  • About 500 ng of the NoTE2 gene expression cassette amplified above was mixed with the host cells, and electroporation was performed under the conditions of 50 ⁇ F, 500 ⁇ , and 2,200 v / 2 mm.
  • the mixture was applied to a 2 ⁇ g / mL zeocin-containing f / 2 agar medium, and cultured at 25 ° C. in a 0.3% CO 2 atmosphere under 12 h / 12 h light / dark conditions for 2 to 3 weeks.
  • those containing the NoTE2 gene expression cassette were selected by PCR.
  • N15P5 medium a nitrogen concentration of f / 2 medium increased 15 times and a phosphorus concentration increased 5 times.
  • the seed culture solution was cultured under shaking at 25 ° C. in a 0.3% CO 2 atmosphere for 12 weeks under 12 h / 12 h light / dark conditions.
  • 2 mL of the seed culture solution was transferred to 18 mL of N15P5 medium, and cultured with shaking for 1 week in a 12 h / 12 h light / dark condition at 25 ° C. in a 0.3% CO 2 atmosphere.
  • Nitrogen gas was blown onto the resulting chloroform layer to dry it, 0.7 mL of 0.5N potassium hydroxide / methanol solution was added, and the temperature was kept constant at 80 ° C. for 30 minutes. Subsequently, 1 mL of 14% boron trifluoride methanol solution (manufactured by SIGMA) was added, and the temperature was kept constant at 80 ° C. for 10 minutes. Thereafter, 0.5 mL of hexane and 1 mL of saturated saline were added and stirred vigorously, and allowed to stand at room temperature for 10 minutes. The upper hexane layer was recovered to obtain a fatty acid methyl ester.

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Abstract

L'invention vise à fournir un procédé de production d'un lipide, ledit procédé améliorant la productivité d'un acide gras à chaîne moyenne ou d'un lipide ayant ledit acide gras en tant que constituant. L'invention concerne un procédé de production d'un lipide, moyennant quoi un transformant, qui est obtenu par l'introduction, dans un hôte, d'un gène codant pour l'une quelconque des protéines (A) à (C) mentionnées ci-dessous, est cultivé, et un lipide est recueilli à partir de la culture. (A) Une protéine qui comprend une séquence du 91ième au 348ième acide aminé de SEQ ID NO: 1. (B) Une protéine qui comprend une séquence d'acides aminés ayant au moins 80 % d'identité avec une séquence du 91ième au 348ième acide aminé de SEQ ID NO : 1, et présente une activité acyl-ACP thioestérase. (C) Une protéine qui présente une séquence d'acides aminés de la protéine (A) ou (B) et présente une activité acyl-ACP thioestérase.
PCT/JP2015/081356 2014-11-14 2015-11-06 Procédé de production de lipide à l'aide d'acyl-acp thioestérase WO2016076231A1 (fr)

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MYPI2017000570A MY182519A (en) 2014-11-14 2015-11-06 Method for producing lipid using acyl-acp thioesterase
US15/520,138 US10280440B2 (en) 2014-11-14 2015-11-06 Method of producing lipid using acyl-ACP thioesterase
JP2016559023A JP6629749B2 (ja) 2014-11-14 2015-11-06 アシル−ａｃｐチオエステラーゼを用いた脂質の製造方法

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WO2018105420A1 (fr) * 2016-12-07 2018-06-14 花王株式会社 Méthode de production de transformé
US10066248B2 (en) 2014-03-03 2018-09-04 Kao Corporation Method of producing lipid by using β-ketoacyl-ACP synthase
US10087428B2 (en) 2012-12-27 2018-10-02 Kao Corporation Acyl-ACP thioesterase
US10508292B2 (en) 2015-05-22 2019-12-17 Kao Corporation Method of producing lipid
US10550412B2 (en) 2014-06-20 2020-02-04 Kao Corporation Method of producing lipid

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Cited By (9)

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US10087428B2 (en) 2012-12-27 2018-10-02 Kao Corporation Acyl-ACP thioesterase
US10597646B2 (en) 2012-12-27 2020-03-24 Kao Corporation Acyl-ACP thioesterase
US10066248B2 (en) 2014-03-03 2018-09-04 Kao Corporation Method of producing lipid by using β-ketoacyl-ACP synthase
US10550412B2 (en) 2014-06-20 2020-02-04 Kao Corporation Method of producing lipid
US10508292B2 (en) 2015-05-22 2019-12-17 Kao Corporation Method of producing lipid
JP2017209101A (ja) * 2016-05-24 2017-11-30 花王株式会社 藻類の処理方法
WO2018105420A1 (fr) * 2016-12-07 2018-06-14 花王株式会社 Méthode de production de transformé
JP2018088902A (ja) * 2016-12-07 2018-06-14 花王株式会社 相同組換えが生じた形質転換体の取得確率を向上させる方法
US11306320B2 (en) 2016-12-07 2022-04-19 Kao Corporation Method for promoting homologous recombination

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